Osteocytes are three-dimensional (3D) ellipsoidal shaped mature bone cells encased in mineralized extracellular matrix. Abundant evidence has shown that the osteocytes are the key mechanonsensor cells that directly regulate bone-forming osteoblast and bone-removing osteoclast activities. Thus, osteocyte functions are critical to the etiology and treatments of osteoporosis, one of the most challenging health issues in the U.S. The proposed project will develop a pseudo-3 dimensional (3D) real-time microscopy technology to simultaneously visualize cell deformation, actin and microtubule cytoskeleton dynamics, and mechano- signaling activation in real-time using biosensors of osteocyte cells under dynamic fluid flow. The proposed technology also incorporates advanced computational fluid dynamics (CFD)-solid modeling of a cell under fluid flow for the estimation of "real-time" cell viscoelastic properties. Given the dynamic nature of fluid flow, cell deformation, and the timescale of mechanically-induced Src/FAK activation (<0.3 seconds), any imaging technique must have a temporal resolution capable of capturing cell deformation at these timescales. Here, we propose using a real-time "pseudo-3D" technique to image two orthogonal views of a cell simultaneously to greater capture the spatial dynamics of actin and microtubule network deformation, Src or FAK activation, and whole cell mechanical properties of micropatterned, ellipsoidally shaped osteocytes under oscillatory flow. The specific aims of the project are: (1) Simultaneously track and analyze individual cell deformation, actin filament networks, and microtubule networks using pseudo-3D imaging of osteocytes under oscillatory fluid flow and correlate the predicted whole cell mechanical properties with the intracellular actin filament or microtubule network strains and (2) Simultaneously track intracellular actin or microtubule network deformation and Src kinase or FAK activation by fluorescence resonance energy transfer (FRET) biosensors using pseudo-3D imaging of osteocytes under oscillatory fluid flow and correlate the localized biosensor activation with the network strains. This study will have significant impact in the basic science research in the field of bone biology, cellular mechanotransduction, cell adhesion, and the mechanobiology of many adherent cell types. PUBLIC HEALTH RELEVANCE: This R21 application will develop a real-time "pseudo-3D" microscopy technique to image two orthogonal views of a cell under dynamic fluid flow simultaneously to greater capture the spatial dynamics of cytoskeletal deformation, Src or FAK activation, and whole cell mechanical properties of osteocytes. This enabling technology will have great impact in the general fields of bone biology and cellular mechanics for an increased understanding of the role that physical forces play in bone cell mechanotransduction.